MATERNAL EFFECTS ON OOCYTE QUALITY IN FARMED ATLANTIC HALIBUT (HIPPOGLOSSUS HIPPOGLOSSUS L.) Maren Mommens A Thesis Submitted for the Degree of PhD at the University of St Andrews 2012 Full metadata for this item is available in Research@StAndrews:FullText at: http://research-repository.st-andrews.ac.uk/ Please use this identifier to cite or link to this item: http://hdl.handle.net/10023/3661 This item is protected by original copyright Maternal effects on oocyte quality in farmed Atlantic halibut (Hippoglossus hippoglossus L.) Maren Mommens This thesis is submitted in partial fulfilment for the degree of PhD at the University of St Andrews September 2011 This thesis is dedicated to my parents. 2 Acknowledgements First of all, I would like to express my gratitude to my supervisors Professor Ian A. Johnston, Professor Igor Babiak and Dr. Jorge M.O. Fernandes for their invaluable support, advice and constructive feedback. My thanks go to Risør Fisk AS and especially Kjell E. Naas and Yngve Attramadal for their cooperation and help during sampling. The microarray collaboration with Dr. Knut Erik Tollefsen from the Norwegian Institute for Water Research (NIVA) was much appreciated and I would like to thank You Song for his excellent training in microarray preparation. The members of the Fish Muscle Research Group at the University of St. Andrews introduced me to molecular biology and I would especially like to thank Dr. Hung-Tai Lee and Dr. Sitheswarab Nainamalai for help and support in the lab. Dr. Neil Bower arrived at the same time as me in St.Andrews and together with his family we shared our Scotland experience. Special thanks go to Dr. Lara Meischke for including me in her group of friends which contributed to many memorable experiences during my stay. I would like to thank all members of the Reproductive Biology Group at the University of Nordland for their support and encouragement during my PhD. Special thanks goes to Dr. Sylvie L. Bolla for invaluable help during sampling and sharing her experience and enthusiasm on Atlantic halibut production and fish nutrition with me. Carlos F.C. Lanes and Teshome Bizuayehu contributed to part of this study with samples and help during lab work. I would like to thank Professor Christel Solberg and Dr. Ørjan Hagen from the Seafood Quality Research Group for their contribution to the reference gene analysis. Thanks go to Bjørnar Eggen and Tormod Skålsvik from the Research Station at the University of Nordland for their help during Atlantic halibut stripping. Friends and colleagues at the Faculty of 3 Bioscience and Aquaculture at the University of Nordland helped out by hiring me during the last part of my PhD to make sure that I could finish. During my PhD I was lucky to get to know many lovely people from all over the world, making this time not only a scientific, but also a cultural experience. Their friendships and never ending encouragements are much appreciated. Finally, my greatest gratitude goes to my parents and family for their love, encouragement and support. 4 Table of contents Acknowledgement……………………………………...……………………………………...3 Table of contents…………………………...………………………………………………......5 List of figures…………………………………………………………………………………10 List of tables……………………………….………………………………………………….12 Declaration…………………………………...……………………………………………….14 List of abbreviations…………………..……………………………………………..………..15 Thesis abstract……………………………….……………………………………….……….19 1 General Introduction ........................................................................................................ 21 1.1 Atlantic halibut .......................................................................................................... 21 1.1.1 Atlantic halibut farming and its challenges ........................................................ 22 1.1.2 Atlantic halibut embryonic development ........................................................... 25 1.2 Oocyte quality in teleosts .......................................................................................... 30 1.2.1 Broodstock husbandry ........................................................................................ 30 1.2.2 Oocyte fertilisation and incubation .................................................................... 33 1.2.3 Markers for oocyte quality ................................................................................. 33 1.3 Gene regulation of embryonic development ............................................................. 35 1.3.1 Maternal mRNAs ............................................................................................... 35 1.4 Teleosts genomic resources ....................................................................................... 40 1.4.1 Introduction to teleosts genomes ........................................................................ 40 5 1.4.2 Applications for commercial farming ................................................................ 45 1.4.3 Molecular markers for oocyte quality ................................................................ 48 1.5 Objectives of the present study .................................................................................. 49 2 Construction of a maternal EST library by suppressive subtractive hybridisation (SSH) in Atlantic halibut ......................................................................................................................... 50 2.1 Abstract ...................................................................................................................... 50 2.2 Introduction ............................................................................................................... 51 2.3 Material and Methods ................................................................................................ 53 2.3.1 Fish husbandry and sample collection................................................................ 53 2.3.2 RNA extraction and cDNA synthesis ................................................................. 53 2.3.3 Suppressive subtractive hybridisation ................................................................ 54 2.3.4 Sequence processing and bioinformatics analysis.............................................. 57 2.3.5 Relative gene expression by quantitative real-time PCR (qPCR) ...................... 59 2.3.6 Data analysis and statistics ................................................................................. 60 2.4 Results ....................................................................................................................... 67 2.4.1 Characterization of EST library ......................................................................... 67 2.4.2 Screening of relative gene expression during embryonic development ............. 72 2.5 Discussion .................................................................................................................. 76 2.5.1 Characterization of the EST library.................................................................... 76 2.5.2 Screening of relative gene expression through embryonic development ........... 77 3 Selection of suitable reference genes for quantitative real-time (qPCR) studies of Atlantic halibut development ................................................................................................................. 80 6 3.1 Abstract ...................................................................................................................... 80 3.2 Introduction ............................................................................................................... 81 3.3 Material and Methods ................................................................................................ 85 3.3.1 Fish husbandry and sample collection................................................................ 85 3.3.2 RNA extraction and cDNA synthesis ................................................................. 87 3.3.3 Primer design ...................................................................................................... 87 3.3.4 Quantitative real-time PCR (qPCR) ................................................................... 90 3.3.5 Statistical analysis .............................................................................................. 91 3.3.6 Evaluation of expression stability ...................................................................... 91 3.4 Results ....................................................................................................................... 93 3.4.1 Developmental expression profiles of candidate reference genes ..................... 93 3.4.2 Analysis of expression stability .......................................................................... 93 3.5 Discussion ................................................................................................................ 103 4 Maternal transcripts in Atlantic halibut during embryonic development ...................... 107 4.1 Abstract .................................................................................................................... 107 4.1.1 Fish husbandry and sample collection.............................................................. 111 4.1.2 RNA extraction and cDNA synthesis ............................................................... 112 4.1.3 Relative gene expression by quantitative-real time PCR (qPCR) .................... 112 4.1.4 Data analysis and Statistics .............................................................................